Method and compositions for detecting epidermal growth factor receptor variant forms in cancer cells

ABSTRACT

Method and compositions for screening for the presence of Epidermal Growth Factor Receptor variant 3 (EGFR(v3)) in a sample are described. The method comprises obtaining a sample containing a plurality of cells; hybridizing a set of chromosomal probes to the sample, wherein the set comprises an EGFR(v3)-probe and a probe to chromosome 7 different from an EGFR(v3)-probe; and visualizing the hybridization pattern of the set of chromosomal probes in the plurality of cells of the sample, wherein the presence of at least one copy of chromosome 7 lacking a hybridization signal of the EGFR(v3)-probe in at least one cell is indicative of the presence of the EGFR(v3) in the sample. The method and compositions are suitable for diagnosing the therapeutic outcome for treating a patient having a cancer with an anti-EGFR therapeutic agent and for screening a sample for a predisposition for forming an EGFR-associated cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority under 35 U.S.C. 119 to U.S.provisional patent application Ser. No. 61/582,103, filed Dec. 30, 2011,and entitled “METHOD AND COMPOSITIONS FOR DETECTING EPIDERMAL GROWTHFACTOR RECEPTOR VARIANT FORMS IN CANCER CELLS,” and to U.S. provisionalpatent application Ser. No. 61/582,135, filed Dec. 30, 2011, andentitled “NUCLEIC ACID HYBRIDIZATION PROBES,” to Russell et al., Thecontents of both these applications are herein incorporated by referencein their entireties.

BACKGROUND OF THE INVENTION

Epidermal Growth Factor Receptor (EGFR) is a cell surface protein thatbinds to its ligand, epidermal growth factor (EGF) and related ligands,and controls cell growth and differentiation as a result of thisreceptor-ligand interaction. EGFR is a member of the tyrosine kinasesuperfamily of signaling molecules that participate in ligand-mediated,cell proliferation mechanisms. Like other members of the receptortyrosine kinase family, EGFR is an oncogene that has an important rolein tumorigenesis. Mutations in EGFR are associated with a variety ofcancers, including, for example, adenocarcinoma, adrenocortical cancer,biliary cancer, breast cancer, cervical cancer, colorectal cancer,esophageal cancer, gall bladder cancer, gastric cancer, glioma,glioblastoma, and glioblastoma multiforme, head and neck cancer, lungcancer, pancreatic cancer, and salivary cancer.

Because of its importance in cell proliferation and its involvement asan oncogenic agent in cancers, considerable research interest hasfocused on understanding the mechanism of EGFR activation and signaling.The protein encoded by the EGFR gene is a transmembrane glycoproteinthat possesses an extracellular ligand binding domain, a transmembranedomain, and an intracellular kinase domain. Upon binding to EGF orstructurally related EGF-like molecules, EGFR undergoes proteindimerization and tyrosine autophosphorylation by the intracellularkinase domain that leads to receptor activation. Activated EGFRphosphorylates other cytoplasmic substrates as part of the signalingcascade, resulting in numerous changes in gene expression and cellularphysiology, including anti-apoptosis and increased cell proliferation.

Mutations in the EGFR gene can lead to overexpression and/orinappropriate activation of the EGFR protein and potentially uncheckedcell proliferation. A significant percentage of epithelial cancers areassociated with mutation, rearrangement, and/or ectopic regulation ofthe EGFR gene. In addition, amplification of the EGFR gene occurs inmany cancers. For example, EGFR gene amplification arises in at least40% of malignant gliomas.

Although a number of oncogenic point mutations occur in the EGFR gene,the constitutively active deletion mutation known as EGFR variant III(EGFR(v3)) appears to be the most common activating mutation found todate. For example, EGFR(v3) is the most common deletion mutant expressedin malignant gliomas and has been reported to occur in almost half ofsuch tumors. EGFR(v3) contains an in-frame deletion of exons 2 through 7corresponding to amino acids 6 through 273. This gene produces a 140- to145-kDa receptor with unique epitopes. Deletion of this codinginformation maps to the extracellular ligand-binding domain of EGFR(v3)and impacts the binding affinity of EGFR(v3) for EGF and relatedligands. Although EGFR(v3) cannot bind ligand with high affinity, it isconstitutively autophosphorylated. Consequently, EGFR(v3) is ofsubstantial interest due to its effects on signal transduction and as apotential tumor-specific target.

EGFR is an attractive target for anticancer therapeutic agentdevelopment. EGFR gene amplification status in a given tumor mayindicate whether cancer cells will respond to therapeutics directed toEGFR. And many of the tumors harboring an amplified EGFR gene produce amutant EGFR protein. Thus, prognosis and therapeutic response may alsodepend upon whether the expressed EGFR gene is mutant or wild type.

In situ methods provide a powerful and sensitive means for detecting thepresence of tumor-specific antigens in biological specimens. Severalchallenges remain for detecting in situ the presence of the EGFR(v3)gene products in tumors. The EGFR(v3) mRNA is difficult to detect insitu with reverse transcriptase-based PCR techniques, owing to thehighly degraded state of RNA in formalin-fixed paraffin-embeddedspecimens. Antibodies directed against the EGFR(v3) protein are alsopoor for use in immunohistochemistry methodologies. Thus, the existingin situ methods are not amenable for efficiently detecting EGFR(v3)status in tumors.

SUMMARY

In a first aspect, the invention is a method of screening for thepresence of EGFR(v3) in a sample. The method comprises obtaining asample containing a plurality of cells; hybridizing a set of chromosomalprobes to the sample, wherein the set comprises an EGFR(v3)-probe and aprobe to chromosome 7 different from an EGFR(v3)-probe; and visualizingthe hybridization pattern of the set of chromosomal probes in theplurality of cells of the sample. According to this aspect of theinvention, the presence of at least one copy of chromosome 7 lacking ahybridization signal of the EGFR(v3)-probe in at least one cell isindicative of the presence of the EGFR(v3) in the sample.

In a second aspect, the invention is a method of diagnosing thetherapeutic outcome for treating a patient having a cancer with ananti-EGFR therapeutic agent. The method comprises obtaining a samplecontaining a plurality of cancer cells; hybridizing a set of chromosomalprobes to the sample, wherein the set comprises an EGFR(v3)-probe and aprobe to chromosome 7 different from an EGFR(v3)-probe; and visualizingthe hybridization pattern of the set of chromosomal probes in theplurality of cancer cells in the sample. According to this aspect of theinvention, the presence of at least one copy of chromosome 7 lacking ahybridization signal of the EGFR(v3)-probe in at least one cancer cellis indicative of the cancer having EGFR(v3), wherein the cancer ispredisposed to developing a resistance to treatment with the anti-EGFRtherapeutic agent.

In a third aspect, the invention is a method of screening a sample for apredisposition for forming an EGFR-associated cancer. The methodcomprises obtaining a sample containing a plurality of cells;hybridizing a chromosomal probe to the sample, wherein the chromosomalprobe is at least one member selected from the group consisting of achromosome enumeration probe to chromosome 7, a chromosome arm probe toat least one arm of chromosome 7, and a locus specific probe to a geneor region of chromosome 7; and visualizing the hybridization pattern ofthe chromosomal probe in the plurality of cells of the sample. Accordingto this aspect of the invention, the presence of three or morehybridization signals directed against chromosome 7 in at least one cellis indicative of amplification of chromosome 7 and of the sample beingpredisposed to forming an EGFR-associated cancer.

In a fourth aspect, the invention is a kit consisting of a set ofchromosomal probes and optionally one or more reagents selected from thegroup consisting of a slide, phosphate buffered saline, hybridizationbuffer, 4,6-diamidino-2-phenylindole dihydrochloride, sodiumchloride-sodium citrate solution, fixative, ethanol, non-ionicdetergent, and denaturation buffer. According to this aspect of theinvention, the set of chromosomal probes comprises an EGFR(v3) probe andat least one member selected from the group consisting of a CEP 7 probe,a 7p12 probe and an EGFR probe. Furthermore, the probes are labeled suchthat each probe can be distinctly visualized after hybridization to abiological sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a schematic of the EGFR gene in the region of thedeletion for EGFR(v3) and the amplicons selected for FISH analysis. Theexons are denoted as solid vertical blue bars and are labeled exons 1-7.Amplicons are denoted by a number (e.g., Amplicon 1, Amplicon 2,Amplicon 8, etc.). The following SEQ ID NOs. were used to amplify thefollowing amplicons: Amplicon 1: SEQ. ID NO:1 (1-Forward (EGFR72397F))and SEQ ID NO:2 (1-Reverse (EGFR86421R)); Amplicon 2: SEQ. ID NO:3(2-Forward (EGFR86450F)) and SEQ ID NO:4 (2-Reverse (EGFR100464R));Amplicon 3: SEQ. ID NO: 5 (3-Forward (EGFR100469F)) and SEQ ID NO:6(3-Reverse (EGFR114486R)); Amplicon 4: SEQ. ID NO:7 (4-Forward(EGFR114643F)) and SEQ ID NO:8 (4-Reverse (EGFR128664R)); Amplicon 5:SEQ. ID NO:9 (5-Forward (EGFR128704F)) and SEQ ID NO:10 (5-Reverse(EGFR142703R)); Amplicon 6: SEQ. ID NO:11 (6-Forward (EGFR129278F)) andSEQ ID NO:12 (6-Reverse (EGFR143287R)); Amplicon 7 (SEQ. ID NO:13(7-Forward (EGFR130321F)) and SEQ ID NO:14 (7-Reverse (EGFR144330R));and Amplicon 8 (SEQ. ID NO:9 (8-Forward (EGFR128704F)) and SEQ ID NO:14(8-Reverse (EGFR144330R)).

DETAILED DESCRIPTION

The invention provides improved in situ methods for detecting amplifiedEGFR genes in biological samples for the purpose of detecting EGFR(v3)in EGFR-associated cancers and screening subjects at risk for thesecancers. The described methods also have utility for monitoring patientsdiagnosed with cancer, for assessing prognosis of an effectivetherapeutic response directed against cancers containing EGFR deletionmutations, particularly EGFR(v3). For example, the methods enable aphysician to diagnose the therapeutic outcome for treating a patienthaving a cancer with an anti-EGFR therapeutic agent, particularly forcancers that are predisposed to developing resistance to anti-EGFRtherapeutic agents. The disclosed methods also have utility formonitoring patients diagnosed with cancer and for evaluating tumorrecurrence.

As used herein, “an EFGR-associated cancer” is a cancer comprising cellshaving increased expression of EGFR, possessing amplified copies ofchromosome 7, and/or encoding a genetic alteration of the chromosomallocus encoding EGFR. Cells of such cancers display enhancedproliferation rates in the presence of EGF and EGF-related ligands andpossess elevated levels of gene products that are regulated byEGFR-mediated signaling activities.

The insight recognized by the inventors was that chromosomal DNA may beprobed directly using in situ methods to detect the presence ofamplified and/or deletion mutations of EGFR without having to resort toindependent evaluation of EGFR gene product status at the mRNA orprotein levels. The disclosed methods are simple and robust and providea reproducible, sensitive indication of the EGFR gene status in cells.

In general, a set of chromosomal probes is hybridized to the chromosomesof cells from a biological sample on a slide. The resultanthybridization pattern produced in the biological sample is thenvisualized using suitable cytogenetic techniques, wherein theamplification status of EGFR and the presence of EGFR(v3) may bediscerned.

In Situ Hybridization

The presence or absence of cells containing amplified EGFR and/orEGFR(v3) is determined by in situ hybridization. In general, in situhybridization includes the steps of fixing a biological sample,hybridizing a chromosomal probe to target DNA contained within the fixedbiological sample, washing to remove non-specific binding, and detectingthe hybridized probe.

The EGFR gene spans 186 kbp on chromosome 7 at cytogenetic band 7p12(Entrez Gene reference system). As explained previously, EGFR(v3) is anin-frame deletion variant of the wild-type EGFR gene (“EGFR(wt)”) thatlacks exons 2-7, including the intervening introns contained betweenexons 2 and 7 (that is, introns ##2-6). Removal of substantial portionsof intron 1 and intron 7 often accompanies deletion of exons 2-7.Accordingly, the presence of EGFR(v3) in the cell may be discerned bywhether 12 kbp-135 kbp of unique chromosomal DNA is present or absent atcytogenetic band 7p12 of chromosome 7.

A “biological sample” is a sample that contains cells or cellularmaterial. Typically, the biological sample is concentrated prior tohybridization to increase cell density. Non-limiting examples ofbiological samples include urine, blood, cerebrospinal fluid (CSF),pleural fluid, sputum, and peritoneal fluid, bladder washings,secretions (for example, breast secretion), oral washings, tissuesamples (for example, a biopsy), touch preps, or fine-needle aspirates.The type of biological sample that is used in the methods describedherein depends on the type of cancer one wishes to detect. For example,urine and bladder washings provide useful biological samples for thedetection of bladder cancer and to a lesser extent prostate or kidneycancer. Pleural fluid is useful for detecting lung cancer, mesotheliomaor metastatic tumors (for example, breast cancer), and blood is a usefulbiological sample for detecting leukemia. A biopsy is useful fordetecting gliomas. For tissue samples, the tissue can be fixed andplaced in paraffin for sectioning, or frozen and cut into thin sections.Optionally, tissue samples can be dispersed into single cells by trypsintreatment or any other means of disrupting extracellular connectionsbetween cells.

Cells are typically harvested from a biological sample using standardtechniques. For example, cells can be harvested by centrifuging abiological sample to collect the cells as a pellet.

The cells of the pellet are usually washed in phosphate-buffered saline(PBS). The cells are suspended in PBS and re-collected bycentrifugation. The cells can be fixed, for example, in acid alcoholsolutions, acid acetone solutions, or aldehydes such as formaldehyde,paraformaldehyde, and glutaraldehyde. For example, a fixative containingmethanol and glacial acetic acid in a 3:1 ratio, respectively, can beused as a fixative. A neutral buffered formalin solution also can beused, and includes approximately 1% to 10% of 37-40% formaldehyde in anaqueous solution of sodium phosphate. Slides containing the cells can beprepared by removing a majority of the fixative, leaving theconcentrated cells suspended in only a portion of the solution.

The cell suspension is applied to slides such that the cells do notoverlap on the slide. Cell density can be measured by a light or phasecontrast microscope. For example, cells harvested from a 20 to 100 mlurine sample typically are suspended in a final volume of about 100 to200 μl of fixative. Three volumes of this suspension (usually 3, 10, and30 μl), are then dropped into 6 mm wells of a slide. The density ofcells in these wells is then assessed with a phase contrast microscope.If the well containing the greatest volume of cell suspension does nothave enough cells, the cell suspension is concentrated and placed inanother well.

Prior to in situ hybridization, chromosomal probes and chromosomal DNAcontained within the cell each are denatured. Denaturation process isperformed in several ways. For example, denaturation can be effectedwith buffered solutions having elevated pH, with elevated temperatures(for example, temperatures from about 70° C. to about 95° C.), or withorganic solvents such as formamide and tetraalkylammonium halides, orcombinations thereof. For example, chromosomal DNA can be denatured by acombination of temperatures above 70° C. (for example, about 73° C.) anda denaturation buffer containing 70% formamide and 2×SSC (0.3M sodiumchloride and 0.03 M sodium citrate). Denaturation conditions typicallyare established such that cell morphology is preserved. Chromosomalprobes can be denatured by heat. For example, probes can be heated toabout 73° C. for about five minutes.

After removal of denaturing chemicals or conditions, probes are annealedto the chromosomal DNA under hybridizing conditions. “Hybridizingconditions” are conditions that facilitate annealing between a probe andtarget chromosomal DNA. Hybridization conditions vary, depending on theconcentrations, base compositions, complexities, and lengths of theprobes, as well as salt concentrations, temperatures, and length ofincubation. The greater the concentration of probe, the greater theprobability of forming a hybrid. For example, in situ hybridizations aretypically performed in hybridization buffer containing 1-2×SSC, 50%formamide and blocking DNA to suppress non-specific hybridization. Ingeneral, hybridization conditions, as described above, includetemperatures of about 25° C. to about 55° C., and incubation lengths ofabout 0.5 hours to about 96 hours. More particularly, hybridization canbe performed at about 32° C. to about 40° C. for about 2 to about 16hours.

Non-specific binding of chromosomal probes to DNA outside of the targetregion can be removed by a series of washes. Temperature andconcentration of salt in each wash depend on the desired stringency. Forexample, for high stringency conditions, washes can be carried out atabout 65° C. to about 80° C., using 0.2×SSC to about 2×SSC, and about0.1% to about 1% of a non-ionic detergent such as Nonidet P-40 (NP40).Stringency can be lowered by decreasing the temperature of the washes orby increasing the concentration of salt in the washes.

Chromosomal Probes

Suitable probes for use in the in situ hybridization methods utilizedwith the invention fall into two broad groups: chromosome enumerationprobes and locus-specific probes. Chromosomal enumeration probes areprobes that hybridize to a chromosomal region, usually a repeat sequenceregion, and indicate the presence or absence of an entire chromosome.Locus-specific probes are probes that hybridize to a specific locus on achromosome and detect the presence or absence of a specific locus.Chromosome arm probes are probes that fall between these two broadgroups. The probes belonging to this category hybridize to a chromosomalregion and indicate the presence or absence of an arm of a specificchromosome. For example, a 7p chromosomal arm probe comprises a sequenceisolated from the region between the telomere of the p arm of chromosome7 and the centromere of chromosome 7.

As is well known in the art, a chromosome enumeration probe canhybridize to a repetitive sequence, located either near or removed froma centromere, or can hybridize to a unique sequence located at anyposition on a chromosome. For example, a chromosome enumeration probecan hybridize with repetitive DNA associated with the centromere of achromosome. Centromeres of primate chromosomes contain a complex familyof long tandem repeats of DNA comprised of a monomer repeat length ofabout 171 base pairs that are referred to as alpha-satellite DNA. Todetect amplification of chromosome 7 according the present invention,preferred target sequences include the repetitive DNA associated withthe centromere of this chromosome. Thus, preferred hybridization probesinclude chromosome enumeration probes that hybridize to centromeric DNAof chromosome 7. Examples of chromosome enumeration probes are describedin the Tables and Examples.

A locus-specific probe hybridizes to a specific, non-repetitive geneticlocus on a chromosome. Preferred locus-specific probes hybridize to the7p12 cytogenetic band region of chromosome 7. Even more preferablelocus-specific probes include EGFR gene sequences within the 7p12cytogenetic band region. Specifically, highly preferred locus-specificprobes include those that hybridize to target chromosomal DNA common toboth EGFR(wt) and EGFR(v3). Such highly preferred locus-specific probesinclude probes that hybridize to target chromosomal DNA of the EGFR geneat any of exon 1, exons 8 through 28, introns 8 through 27, and/or theflanking DNA sequences lying immediately 5′ and/or 3′ of the EGFR gene(that is, upstream of exon 1 and/or downstream of exon 28).

To discern the deletion mutation status of EGFR of chromosome 7 as beingEGFR(v3), preferred locus-specific probes are selected, wherein suchprobes hybridize to one or more EGFR-specific DNA sequences at any ofexons 2, 3, 4, 5, 6 and 7, introns 2, 3, 4, 5, and 6, or combinationsthereof. These particular DNA sequences are preferred for use as probesin this regard because these sequences are present in EGFR(wt) andabsent in EGFR(v3). Thus, the status of EGFR(v3) in a cell can bediscerned by the relative absence of a hybridization signal onchromosome 7 when contacted with locus-specific probes directed to thedeleted sequences of EGFR(v3).

As described above, sequences associated with portions of intron 1 andintron 7 of EGFR(wt) are also deleted in EGFR(v3). These deleted intronsequences are also suitable for use as chromosomal probes of the presentinvention. Example 1 provides an illustration of preferred chromosomalprobes directed to deleted sequences of EGFR(v3) that fall within theseintronic sequences.

In other preferred embodiments, locus-specific probes may comprise aplurality of discrete DNA sequences from a particular genetic locus. Forexample, a probe specific for one or more of any of the deleted exons orintrons of EGFR(v3) can comprise a plurality of discrete DNA sequences,wherein each DNA sequence hybridizes to one or more EGFR-specific DNAsequences at any of exons 2, 3, 4, 5, 6 and 7, introns 2, 3, 4, 5, and6, or combinations thereof and wherein each DNA sequence has a length ofat least 50 base-pairs.

Chromosomal probes are chosen for maximal sensitivity and specificity.Using a set of chromosomal probes (that is, two or more probes) providesgreater sensitivity and specificity than use of any one chromosomalprobe. Thus, chromosomal probes that detect both EGFR gene amplificationstatus and the presence of EGFR(v3) are included in a set. For example,a set of chromosomal probes can include chromosome enumeration probe tochromosome 7 (for example, a CEP 7 probe) and a locus-specific probespecific for one or more of any of the deleted exons or introns ofEGFR(v3) (“EGFR(v3)-probe”). Alternatively, a set of chromosomal probescan include two different locus-specific probes, wherein the firstlocus-specific probe hybridizes to chromosome 7 at a locus other thanEGFR and the second locus-specific probe is an EGFR(v3)-probe.Additionally, the foregoing examples of probe sets can include a thirdprobe specific for one or more chromosomal sequences common to bothEGFR(wt) and EGFR(v3) (“EGFR-probe”). Examples of an EGFR-probe includeany one or more of the following sequences or combinations thereof:5′-flanking sequences immediately upstream of exon 1; exon 1, any ofexons 8 through 28, any of introns 7 through 27; and 3′-flankingsequences immediately downstream of exon 28.

The present invention is directed to measuring amplification ofchromosome 7 and loss of chromosomal sequences within EGFR in biologicalsamples. Such measurements pose unique challenges that the skilledartisan would recognize and control for. For example, it is generallyeasier to measure gain of a target chromosome or chromosome region dueto an amplification event than to measure loss of a target chromosome orchromosome region due to a deletion event. The difficulty in measuringloss of chromosome sequence information is confounded by the possibleoccurrence of failed or poor hybridization in cells that falselysuggests loss. Probe designs that contain overlapping sequence contentfor detecting both retained and deleted sequence information may alsoconfound the analysis by hybridizing to a chromosomal region flanking adeletion that falsely suggests no loss.

The latter problem can be addressed by appropriate probe design. Probesthat are designed to detect a deletion event (“deletion probes”), suchas an EGFR(v3)-probe, are designed not to extend beyond the minimallydeleted region. If too much of the deletion probe extends beyond thedeleted sequence, enough signal may be produced in the hybridizationassay to be falsely counted. For this reason, locus-specific probesdesigned to detect deletions are generally smaller than locus-specificprobes designed to detect gains.

The former problem of failed or poor hybridization can be addressed inseveral ways. One approach is to include in the hybridization assay acontrol probe that provides a positive hybridization signal for thepresence of undeleted regions of the specific locus under study (forexample, an EGFR-probe if EGFR is the locus under study). Wheneverpossible, such control probes are designed to have the length andsequence complexity of the deletion probes. This probe design will aidin yielding comparable hybridization signal intensities for deletionprobes and control probes in samples that contain sequences for bothprobes. Another approach is to include control biological samples knownto contain undeleted locus-specific information so that one can confirmunder comparable hybridization conditions that the deletion probehybridizes appropriately to those samples.

Since deletion probes are usually kept small their signals are not asintense as signals for targets typically gained. This in turn makes itmore likely that real signals from targets being monitored for deletionmay be miscounted. Likewise, some chromosome enumeration probes usuallyprovide brighter signals and hybridize faster than locus-specificprobes. Differences in apparent hybridization signal intensity can becompensated for by choosing appropriate probe design and biologicalsamples, and by pre-calibrating the signal intensity of probeshybridized with control biological samples. These considerations aretaken into account when selecting a probe set and biological samples forpracticing the present invention.

Chromosome enumeration probes and locus-specific probes are availablecommercial vendors. Examples of chromosome enumeration probes tochromosome 7 are presented in the Examples. Alternatively, probes can bemade non-commercially from chromosomal or genomic DNA through standardtechniques. For example, sources of DNA that can be used include genomicDNA, cloned DNA sequences, somatic cell hybrids that contain one, or apart of one, human chromosome along with the normal chromosomecomplement of the host, and chromosomes purified by flow cytometry ormicrodissection. The region of interest can be isolated through cloning,or by site-specific amplification via the polymerase chain reaction(PCR). Probes less than 100-200 nucleotides in length may also beprepared by in vitro chemical synthesis methods.

The probes of the present invention comprise DNA having a preferredlength of 50-10,000 base-pairs. In some preferred embodiments, suchprobes comprise DNA having a preferred length of 100-5,000 base-pairs.In other preferred embodiments, such probes comprise DNA having200-2,500 base-pairs.

The choice of probe length and design is often tied to considerations ofthe manner whereby the probes are labeled with a detectable moiety.Labeling design considerations are selected to maximize the amount oflabel incorporated per unit length of the probe, which is often referredto as the specific activity of the labeled probe. For relatively smallprobes, such as probes less than 200 nucleotides in length or probesthat are prepared by chemical means, chemical or enzymatic end-labelingone or both termini of the probe is a preferred method of labeling. Forexample, carboxytetramethylrhodamine (CTMR) is a preferred chemicallabeling reagent for this purpose. For longer probes, such as probesgreater than 100 nucleotides in length or probes can be prepared byenzymatic amplification means, for example, with the polymerase chainreaction (PCR), a preferred method of labeling is nick-translation orPCR-labeling using labeled deoxynucleotide triphosphates. One skilled inthe art would recognize that these labeling methods are described forillustrative purposes and that any method of incorporating a label intoa nucleic acid would represent an acceptable means of preparing labeledprobes of the present invention.

As explained above, probes that include non-repetitive genetic locusinformation are preferred embodiments of a locus-specific probe. In FISHhybridization applications, it can be important to further refine thetarget sequences to exclude certain repetitive sequence elements foundubiquitously in natural sequences. In this regard, one can select apriori the precise boundaries and composition of the desired targetsequences by virtue of PCR primer design that specifies the PCR productsto be generated. For example, one can analyze a genetic locus orplurality of genetic loci from a given chromosomal region of interest todelineate non-repetitive genetic information (for example, gene-codinginformation) from repetitive sequence elements found dispersedthroughout all chromosomal DNA (for example, SINEs, LINEs, and LTRs).Once delineated, one can then design PCR primers that can be used toamplify only the non-repetitive genetic information (that is, sequenceslacking repetitive sequence elements as defined above) as the preferredtarget sequences. In many cases, the preferred target sequences canrange in size from about 100 bp about 6 kbp. Such non-repetitivesequence material is preferred for use in FISH applications, because theresultant probes generated from non-repetitive sequence element DNAobviates or at least substantially reduces the need to include C_(o)t-1DNA in FISH applications, the latter of which reduces signal intensityand robustness. Once obtained, these non-repetitive sequences can beisolated or purified for use as substrates for preparing PCR-generatedprobes according to the methods outlined above. Related nucleichybridization probes for this purpose can be accomplished using themethods disclosed in U.S. patent application serial number ______, filedsimultaneously to this application, entitled NUCLEIC ACID HYBRIDIZATIONPROBES to Russell et al., which claims benefit of priority to U.S.provisional patent application Ser. No. 61/582,135, filed Dec. 30, 2011,the contents of each application are herein incorporated by reference intheir entireties.

Chromosomal probes typically are directly labeled with a fluorophore, anorganic molecule that fluoresces after absorbing light of lowerwavelength/higher energy. The fluorophore allows the probe to bevisualized without a secondary detection molecule. After covalentlyattaching a fluorophore to a nucleotide, the nucleotide can be directlyincorporated into the probe with standard techniques such as nicktranslation, random priming, and PCR labeling. Alternatively,deoxycytidine nucleotides within the probe can be transaminated with alinker. The fluorophore then is covalently attached to the transaminateddeoxycytidine nucleotides. See, U.S. Pat. No. 5,491,224.

Fluorophores of different colors are chosen such that each chromosomalprobe in the set can be distinctly visualized. For example, acombination of the following fluorophores may be used:7-amino-4-methylcoumarin-3-acetic acid (AMCA), Texas Red™ (MolecularProbes, Inc., Eugene, Oreg.), 5-(and-6)-carboxy-X-rhodamine, lissaminerhodamine B, 5-(and-6)-carboxyfluorescein, fluorescein-5-isothiocyanate(FITC), 7-diethylaminocoumarin-3-carboxylic acid,tetramethylrhodamine-5-(and-6)-isothiocyanate,5-(and-6)-carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylicacid, 6-[fluorescein 5-(and-6)-carboxamido]hexanoic acid,N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3-indacenepropionicacid, eosin-5-isothiocyanate, erythrosin-5-isothiocyanate, and Cascade™blue acetylazide (Molecular Probes, Inc., Eugene, Oreg.).

Probes are visualized with a fluorescence microscope and an appropriatefilter for each fluorophore, or by using dual or triple band-pass filtersets to observe multiple fluorophores. See, for example, U.S. Pat. No.5,776,688. Alternatively, techniques such as flow cytometry can be usedto examine the hybridization pattern of the chromosomal probes.

Probes also can be indirectly labeled with biotin or digoxygenin, orlabeled with radioactive isotopes such as ³²P and ³H, although secondarydetection molecules or further processing then is required to visualizethe probes. For example, a probe indirectly labeled with biotin can bedetected by avidin conjugated to a detectable marker. For example,avidin can be conjugated to an enzymatic marker such as alkalinephosphatase or horseradish peroxidase. Enzymatic markers can be detectedin standard calorimetric reactions using a substrate and/or a catalystfor the enzyme. Catalysts for alkaline phosphatase include5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.Diaminobenzoate can be used as a catalyst for horseradish peroxidase.

According the present invention, a preferred method of detecting thehybridization signals by chromosomal probes in cells of a biologicalsample is in situ fluorescence hybridization (FISH) with a set offluorescently labeled probes. Each probe is distinguished from otherprobes based upon different fluorescent labels attached to the probes.Examples of probe sets are described Example 1.

Fluorescent labels are visualized by any suitable means of detectingfluorescence. Preferably, the labels are visualized with fluorescencemicroscopy and appropriate filters. Because the loss of a specifichybridization signal at the EGFR locus of chromosome 7 is diagnostic forthe presence of EGFR(v3), it is preferable to quantitatively measure theintensity of the hybridization signals with digital imaging. Suchtechniques and automated digital imaging systems are well known in theart.

Selection of Cells

According to the invention, cells are microscopically selected from thecells of a biological sample (for example, urine) on a slide prior tochromosomal analysis. “Selecting” refers to the identification of cellsthat are more likely to be neoplastic due to one or more cytologic(mainly nuclear) abnormalities such as nuclear enlargement, nuclearirregularity or abnormal nuclear staining (usually a mottled stainingpattern). These nuclear features, can be assessed with nucleic acidstains or dyes such as propidium iodide or 4,6-diamidino-2-phenylindoledihydrochloride (DAPI). Propidium iodide is a red-fluorescingDNA-specific dye that can be observed at an emission peak wavelength of614 nm. Typically, propidium iodide is used at a concentration of about0.4 μg/ml to about 5 μg/ml. DAPI, a blue fluorescing DNA-specific stainthat can be observed at an emission peak wavelength of 452 nm, generallyis used at a concentration ranging from about 125 ng/ml to about 1000ng/ml. Staining of cells with DAPI or propidium iodide is generallyperformed after in situ hybridization is performed.

Determining Presence of Cells Having Amplified Chromosome 7

After cells are selected based on one or more of the stated criteria,the presence or absence of chromosome 7 amplification is assessed byexamining the hybridization pattern of the chromosomal probes (that is,the number of signals for each probe) in each selected cell andrecording the number of chromosome signals. Chromosome 7 amplificationwill be revealed by the presence of greater than two hybridizationsignals per cell nucleus when analyzed with a probe set that includes achromosome 7-specific probe (for example, a CEP 7 probe or anylocus-specific probe to a chromosome 7 gene or region). This step isrepeated until the hybridization pattern has been assessed in at least 4cells that contain amplified chromosome 7. In a typical assay, thehybridization pattern is assessed in about 20 to about 25 selectedcells, or a sufficient number of cells to permit an adequate statisticalanalysis of the resultant hybridization data.

Determining Presence of Cells Having EGFR(v3)

Cells with more than two copies of chromosome 7 are consideredpredisposed to having an EGFR mutation. To evaluate whether the cellscontain EGFR(v3), cells are analyzed with a chromosomal probe setcomprising a first probe and a second probe. The first probe comprises achromosome 7-specific probe other than an EGFR(v3)-probe (for example, aCEP 7 probe or an EGFR-probe). The second probe comprises anEGFR(v3)-probe.

The first probe identifies all copies of chromosome 7 present in a givencell regardless of the status of EGFR. The second probe identifieswhether deleted exon information of EGFR(v3) is present or absent in theresident EGFR gene of each chromosome 7 for that cell.

Cells lacking EGFR(v3) have each copy of chromosome 7 hybridized to bothprobes. Cells containing EGFR(v3) have at least one copy of chromosome 7that fails to hybridize to only the EGFR(v3)-probe. Thus, the presenceof EGFR(v3) in a given cell will be discerned by the presence of atleast one copy of chromosome 7 lacking a hybridization signal to onlythe EGFR(v3)-probe.

The sensitivity and accuracy of the foregoing analysis is improved byincluding a third probe directed to a region of chromosome 7 other thanthe centromere. A preferred probe is one that hybridizes to sequences atcytogenetic band 7p12 (“7p12-probe”). A preferred 7p12-probe for thispurpose is an EGFR-probe. Accordingly, the amplification status ofchromosome 7, as well as the presence of sequences associated with EGFR,may be confirmed by detecting an independent hybridization signal withan EGFR-probe.

Monitoring Patients for EGFR-Associated Cancer

The superior sensitivity of the methods described herein is amenable fordetecting and monitoring of cancers associated with amplification ofchromosome 7 and EGFR(v3) status. A patient with a glioma or other typeof cancer may have increased chromosome 7 ploidy (that is, amplificationof chromosome 7 beyond the two copies normally present in a somaticcell) and EGFR(v3). The application of the methods and compositions ofthe present invention provides an effective means to monitor treatmentefficacy directed to EGFR. For example, the method enables the physicianto diagnose the therapeutic outcome for treating a patient having acancer with an anti-EGFR therapeutic agent, particularly for cancersthat are predisposed to developing resistance to anti-EGFR therapeuticagents. The method of the present invention enables a physician toconfirm the presence of EGFR(v3) in cancer cells, thereby permitting thephysician to develop an alternative treatment strategy as warranted. Themethods and compositions of the present invention also enable one tosurvey for tumor recurrence/progression in patients with EGFR-associatedcancers. An EFGR-associated cancer for which the described methods andcompositions of the invention may be used includes adenocarcinoma,adrenocortical cancer, biliary cancer, breast cancer, cervical cancer,colorectal cancer, esophageal cancer, gall bladder cancer, gastriccancer, glioma, glioblastoma, and glioblastoma multiforme, head and neckcancer, lung cancer, pancreatic cancer, and salivary cancer.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Probe Preparation

Primers specific to EGFR were designed for amplification of sequenceencompassing the EGFR variant 3. Primers were designed to have a highT_(M) to promote specificity and with restriction sites at the 5′ endfor cloning of the PCR products. Primers in combination with BAC DNAcontaining EGFR sequences were used to amplify specific regions of theEGFR gene such that one amplicon (#8) covered exons two through seven(see FIG. 1). Other primer combinations were used to amplify successiveregions upstream of amplicon 8 (for example, amplicons 2, 3 and 4; seeFIG. 1). The primers used for amplifying each of the ampliconsillustrated in FIG. 1 are illustrated in Table 1 below. As inferred fromTable 1, amplicon 8 corresponds to the region spanning amplicons 5-7 ofFIG. 1.

TABLE 1 EGFR Amplicon Amplification Primer Sets SEQ SEQUENCE AmpliconID NO. (5′→3′) Target 1 CATCATCCGCGGAAGGATGCACAATCCTACATGCC 1-Forward CC2 CATCATATCGATCCTGGTGAACACAACCGGAGAAT 1-Reverse TAAA 3CGGATCCTGTGTACCCCATGTGTTTAAATTTGCTG 2-Forward 4CAGGTACCTTCTTGGGTTAGCAGTGATCAAGTCAC 2-Reverse A 5CAACTAGTATAAAGCAGAAGCATGTATCCAGGTTG 3-Forward C 6CCTCGAGCAAGCCTTGGCCCAGGCTTATTC 3-Reverse 7CATCATCCGCGGCATGAAATCTGCATTATCATCAT 4-Forward CTGCA 8CATCATATCGATGCATATGGACTTACTCAAATGTT 4-Reverse GGCCA 9CGGATCCTATAGAGTGGCTGACATCCCCTAACGTG 5-Forward 10CCTCGAGACTTGCCACGGCAGCGCC 5-Reverse 11CATCGCGGCCGCACCATTTTCATATTTGAGGAAAG 6-Forward CATGG 12CCTCGAGTGGTGGAGGAGCCGAGGGATC 6-Reverse 13CATCGCGGCCGCTTCTTTGGTTCAGCTGAGAGAAA 7-Forward CTTGC 14CCTCGAGCAGGGCTCAGCAGGAGACAGAGC 7-Reverse 9CGGATCCTATAGAGTGGCTGACATCCCCTAACGTG 8-Forward 14CCTCGAGCAGGGCTCAGCAGGAGACAGAGC 8-Reverse

The amplicons were prepared by PCR using the following PCR conditions.The PCR reaction conditions were performed in 1× Herculase buffercontaining 3% DMSO, 500 μM dNTP, 0.5 μM each primer and 10 U Herculase.The PCR amplification conditions were as follows: (1) 92° C. for 2minutes hot start; (2) ten cycles of: (a) 92° C. for 10 seconds, (b1)64° (amplicons 3 and 4) or (b2) 68° C. (amplicons 2 and 8) for 10minutes; twenty-five cycles of: 92° C. for 10 seconds; (c1) 64°(amplicons 3 and 4) or (c2) 68° C. (amplicons 2 and 8) for 10 minutesplus 10 second for successive cycles; and (3) 68° C. for 7 minutes.

Once amplified, PCR products were either nick-translated or chemicallylabeled with CTMR (providing an orange color). The full-length EGFRprobe was labeled in green using a nick translation kit. The CEP-7 probewas labeled in aqua using a nick translation kit.

FISH Method for Detecting EGFR and EGFR(v3) Status

The probes were used to hybridize to paraffin sections of gliomaspecimens following pretreatment with Hopmann's paraffin pretreatmentreagent.

Hybridization was performed with the HYBrite method or a conventionalmethod. In the HYBrite method, a HYBrite™ system from Abbott Molecular(Downers Grove, Ill.) was used. Slides were placed on the HYBrite, andabout 10 μl of the probe set was added, covered and sealed. The HYBritewas programmed as follows: 73° C. for 5 min, then 37° C. for 16 hr.Slides were washed with 0.4×SSC (0.06 M sodium chloride/0.006 M sodiumcitrate)/0.3% NP-40 at 73° C. for 2 min., rinsed with 2×SSC/0.1% NP40 atroom temperature for 2 min. and air dried. Slides were counter-stainedwith approximately 10 μl of DAPI II (125 ng/ml of4,6-diamidino-2-phenylindole dihydrochloride).

In the conventional method (that is, Coplin jar method), a master mixcontaining chromosome probes was prepared in hybridization buffercontaining 50% formamide, 2×SSC, 0.5 μg/ml Cot1 DNA, and 2 μg/ml HP DNA.The probe mix was denatured at 73° C. for 5 min., and slides weredenatured in denaturation buffer (70% formamide, 2×SSC) in a Coplin jarat 73° C. for 5 min (6-8 slides/jar). Slides were rinsed in each of 70%,85%, and 100% ethanol for 1 min. Approximately 10 μl of hybridizationmix was applied to each slide, covered with a cover slip, and sealedwith rubber cement. Hybridization was performed in a humidified chamberat 37° C. overnight. Slides were washed in 0.4×SSC/0.3% NP-40 at 73° C.for 2 min., then rinsed briefly in 2×SSC/0.1% NP-40 at room temperature.After the slides are air-dried, slides were counter-stained with DAPIII. Samples were enumerated by recording the number of FISH signals in100 consecutive cells.

An aqua filter was used to visualize the CEP 7 probe. Individual greenand orange filters were used to visualize the EGFR-probe andEGFR(v3)-probe, respectively.

The results of the FISH analyses indicated that Amplicons 4 and 8provided the strongest and cleanest signals for detecting the absence orpresence of the EGFR(v3) deletion sequences. FISH analyses wereperformed using these amplicons as single probes or as probe mixtureswhen used in combination with full-length EGFR probe and CEP 7 probe.

Example 2 Samples and Sample Preparation

Samples will include tissue from 20 biopsy proven glioma cancer cases,in which a diagnosis is made by either positive cytology or byhistology, in the case of cytology negative samples. Control sampleswill include ten blood samples from normal healthy donors (age 25-80).

Approximately 50 mg of glioma tissue material will be collected perpatient and treated with trypsin to generate single cell suspensions.Five mL of blood will collected from the healthy donors. The sampleswill be stored at 4° C. for less than 48 hr, and processed bycentrifugation at 1200×g for 5 min. The supernatant will be discarded,and the pellet will be suspended in 10 ml of 0.075 M KCl, and incubatedat room temperature for 15 minutes. Samples will be processed bycentrifugation at 1200×g for 5 min. to remove the KCl solution. Theresultant pellets will be suspended in 10 ml of a 3:1 methanol:glacialacetic acid fixative, and processed by centrifugation at 1200×g for 5min. The fixative will be carefully removed leaving the cell pellet, andthis step will be repeated two more times.

Density of the cells on the slides will be monitored by frequentlychecking the slides between droppings using a phase contrast microscopefitted a 20× power objective. As many cells as possible will be loadedonto the slide without having cell overlap. Slides will be driedovernight at room temperature.

Slides containing the samples will be incubated in 2×SSC at 37° C. for10-30 min. The slides will then be incubated in 0.2 mg/ml pepsin at 37°C. for 20 min. Slides will be subsequently washed twice in PBS at roomtemperature for 2 min. Cells will be fixed in 2.5% Neutral BufferedFormalin at room temperature for 5 min. Slides will be subsequentlywashed twice in PBS at room temperature for 2 min. The slides will besubjected to dehydration by successive contact in solutions of 70%, 85%,and 100% ethanol at room temperature for 1 min. The slides will be usedimmediately thereafter or stored at room temperature in the dark.

FISH Method for Detecting Chromosome 7 Amplification and EGFR

Three multicolor probe sets: A, B, and C will be used in the initialhybridizations. Probe sets A-C will contain thecentromeric/locus-specific probes shown in Table 2. The color of thefluorophore used to label each probe is also shown in Table 2. An aquafilter will be used to visualize the CEP 7 probe. A dual green/redfilter or individual green and orange filters will be used to visualizethe EGFR-probe and EGFR(v3)-probe, respectively. An red filter will beused to visualize the 7p12 probe.

TABLE 2 FISH Probe Sets Probe Spectrum Spectrum Spectrum Spectrum SetAqua Orange Green Red A CEP 7 EGFR(v3) EGFR B CEP 7 EGFR(v3) 12p7 CEGFR(v3) EGFR

Hybridization will be performed with the HYBrite method or aconventional method. In the HYBrite method, a HYBrite™ system fromAbbott Molecular (Downers Grove, Ill.) will be used. Slides will beplaced on the HYBrite, and about 10 μl of the probe set will be added,covered, and sealed. The HYBrite will be programmed as follows: 73° C.for 5 min, then 37° C. for 16 hr. Slides will then be washed with0.4×SSC (0.06 M sodium chloride/0.006 M sodium citrate)/0.3% NP-40 at73° C. for 2 min., rinsed with 2×SSC/0.1% NP40 at room temperature for 2min., and air dried. Slides will be counter-stained with approximately10 μl of DAPI II (125 ng/ml of 4,6-diamidino-2-phenylindoledihydrochloride).

In the conventional method (that is, Coplin jar method), a master mixcontaining chromosome probes will be prepared in hybridization buffercontaining 50% formamide, 2×SSC, 0.5 μg/ml Cot1 DNA, and 2 μg/ml HP DNA.The probe mix will be denatured at 73° C. for 5 min., and slides will bedenatured in denaturation buffer (70% formamide, 2×SSC) in a Coplin jarat 73° C. for 5 min (6-8 slides/jar). Slides will be rinsed in each of70%, 85%, and 100% ethanol for 1 minute. Approximately 10 μl ofhybridization mix will be applied to each slide, covered with a coverslip, and sealed with rubber cement. Hybridization will be performed ina humidified chamber at 37° C. overnight. Slides will be washed in0.4×SSC/0.3% NP-40 at 73° C. for 2 min., then rinsed briefly in2×SSC/0.1% NP-40 at room temperature. After the slides are air-dried,slides will be counter-stained with DAPI II. Samples will be enumeratedby recording the number of FISH signals in 100 consecutive cells.

Other Embodiments

It is understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims. While various embodiments of the invention have beendescribed, it will be apparent to those of ordinary skill in the artthat other embodiments and implementations are possible within the scopeof the invention. Accordingly, the invention is not to be restrictedexcept in light of the attached claims and their equivalents.

1. A method of screening for the presence of EGFR(v3) in a sample, themethod comprising: a. obtaining a sample containing a plurality ofcells; b. hybridizing a set of chromosomal probes to the sample, whereinthe set comprises an EGFR(v3)-probe and a probe to chromosome 7different from an EGFR(v3)-probe; and c. visualizing the hybridizationpattern of the set of chromosomal probes in the plurality of cells ofthe sample, wherein the presence of at least one copy of chromosome 7lacking a hybridization signal of the EGFR(v3)-probe in at least onecell is indicative of the presence of the EGFR(v3) in the sample.
 2. Themethod of claim 1, wherein the sample is selected from the groupconsisting of urine, blood, cerebrospinal fluid, pleural fluid, sputum,peritoneal fluid, bladder washing, secretion, oral washing, tissuesample, touch prep and fine-needle aspirate.
 3. The method of claim 1,wherein the sample comprises a tissue sample.
 4. The method of claim 1,wherein the probe to chromosome 7 different from an EGFR(v3)-probe isselected from the group consisting of a chromosome enumeration probe, achromosome arm probe, and an EGFR-probe.
 5. The method of claim 1,wherein the probe to chromosome 7 different from an EGFR(v3)-probe isselected from the group consisting of a CEP 7 probe and an EGFR-probe.6. The method of claim 1, wherein the set of chromosomal probescomprises at least one nucleic acid sequence lacking repetitive sequenceelements.
 7. The method of claim 1, wherein the set of chromosomalprobes are fluorescently labeled.
 8. The method of claim 1, whereinvisualizing the hybridization pattern of the set of chromosomal probesin the plurality of cells of the sample is performed by fluorescencemicroscopy.
 9. The method of claim 8, wherein fluorescence microscopy isperformed with digital imaging.
 10. A method of diagnosing thetherapeutic outcome for treating a patient having a cancer with ananti-EGFR therapeutic agent, the method comprising: a. obtaining asample containing a plurality of cancer cells; b. hybridizing a set ofchromosomal probes to the sample, wherein the set comprises anEGFR(v3)-probe and a probe to chromosome 7 different from anEGFR(v3)-probe; and c. visualizing the hybridization pattern of the setof chromosomal probes in the plurality of cancer cells in the sample,wherein the presence of at least one copy of chromosome 7 lacking ahybridization signal of the EGFR(v3)-probe in at least one cancer cellis indicative of the cancer having EGFR(v3) and wherein the cancer ispredisposed to developing a resistance to treatment with the anti-EGFRtherapeutic agent.
 11. The method of claim 10, wherein the cancer isselected from the group consisting of adenocarcinoma, adrenocorticalcancer, biliary cancer, breast cancer, cervical cancer, colorectalcancer, esophageal cancer, gall bladder cancer, gastric cancer, glioma,glioblastoma, glioblastoma multiforme, head and neck cancer, lungcancer, pancreatic cancer, and salivary cancer.
 12. The method of claim10, wherein the cancer is selected from the group consisting of glioma,glioblastoma, and glioblastoma multiforme.
 13. The method of claim 10,wherein the sample is selected from the group consisting of urine,blood, cerebrospinal fluid, pleural fluid, sputum, peritoneal fluid,bladder washing, secretion, oral washing, tissue sample, touch prep andfine-needle aspirate.
 14. The method of claim 10, wherein the probe tochromosome 7 different from an EGFR(v3)-probe is selected from the groupconsisting of a chromosome enumeration probe, a chromosome arm probe,and an EGFR-probe.
 15. The method of claim 10, wherein the set ofchromosomal probes comprises at least one nucleic acid sequence lackingrepetitive sequence elements.
 16. The method of claim 10, wherein theset of chromosomal probes are fluorescently labeled.
 17. The method ofclaim 10, wherein visualizing the hybridization pattern of the set ofchromosomal probes in the plurality of cells of the sample is performedby fluorescence microscopy.
 18. The method of claim 17 whereinfluorescence microscopy is performed with digital imaging.
 19. Themethod of claim 10, wherein the sample comprises a tissue sample.
 20. Amethod of screening a sample for a predisposition for forming anEGFR-associated cancer, the method comprising: a. obtaining a samplecontaining a plurality of cells; b. hybridizing a chromosomal probe tothe sample, wherein the chromosomal probe is at least one memberselected from the group consisting of a chromosome enumeration probe tochromosome 7, a chromosome arm probe to at least one arm of chromosome7, and a locus specific probe to a gene or region of chromosome 7; andc. visualizing the hybridization pattern of the chromosomal probe in theplurality of cells of the sample, wherein the presence of three or morehybridization signals directed against chromosome 7 in at least one cellis indicative of amplification of chromosome 7 and of the sample beingpredisposed to forming an EGFR-associated cancer.
 21. The method ofclaim 20, wherein the chromosomal probe is at least one member selectedfrom the group consisting of a CEP 7 probe, a 7p12 probe, and an EGFRprobe.
 22. The method of claim 20, wherein the chromosomal probecomprises at least one nucleic acid sequence lacking repetitive sequenceelements.
 23. The method of claim 20, further comprising: d. hybridizingan EGFR(v3) probe to the sample; and e. visualizing the hybridizationpattern of the EGFR(v3) probe in the plurality of cells of the sample,wherein the presence of at least one copy of chromosome 7 lacking ahybridization signal of the EGFR(v3)-probe in at least one cell isindicative of the presence of the sample containing an EGFR-associatedcancer having EGFR(v3).
 24. The method of claim 23, wherein theEGFR-associated cancer is selected from the group consisting ofadenocarcinoma, adrenocortical cancer, biliary cancer, breast cancer,cervical cancer, colorectal cancer, esophageal cancer, gall bladdercancer, gastric cancer, glioma, glioblastoma, glioblastoma multiforme,head and neck cancer, lung cancer, pancreatic cancer, and salivarycancer.
 25. The method of claim 23, wherein the sample is selected fromthe group consisting of urine, blood, cerebrospinal fluid, pleuralfluid, sputum, peritoneal fluid, bladder washing, secretion, oralwashing, tissue sample, touch prep and fine-needle aspirate.
 26. Themethod of claim 23, wherein the chromosomal probe comprises at least onenucleic acid sequence lacking repetitive sequence elements.
 27. Themethod of claim 23, wherein the chromosomal probe and the EGFR(v3) probeis fluorescently labeled.
 28. The method of claim 23, whereinvisualizing the hybridization pattern of the chromosomal probe and theEGFR(v3) probe in the plurality of cells of the sample is performed byfluorescence microscopy.
 29. The method of claim 28, whereinfluorescence microscopy is performed with digital imaging.
 30. A kitconsisting of a set of chromosomal probes and optionally one or morereagents selected from the group consisting of a slide, phosphatebuffered saline, hybridization buffer, 4,6-diamidino-2-phenylindoledihydrochloride, sodium chloride-sodium citrate solution, fixative,ethanol, non-ionic detergent, and denaturation buffer, wherein the setof chromosomal probes comprises an EGFR(v3) probe and at least onemember selected from the group consisting of a CEP 7 probe, a 7p12 probeand an EGFR probe, wherein the probes are labeled such that each probecan be distinctly visualized after hybridization to a biological sample.31. The kit of claim 30, wherein the set of chromosomal probes comprisesat least one nucleic acid sequence lacking repetitive sequence elements.